Department of Cell Biology and Molecular Genetics, Institute for Physical Science and Technology, Department of Bioengineering, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, United States of America.

Abstract

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.

(1) Gene similarity trees are built around each gene in E. coli, including any distantly related gene in the RAST database. (2) A matrix is formed which links genes with their primary functions and also potential promiscuous functions. A gene (in this example, eco1) will take a potential secondary ‘promiscuous’ function in the matrix if its similarity tree includes any genes annotated with different functions (e.g., in this example, shi4, which encodes function fn4). (3) Cases in which a gene’s predicted promiscuous function is identical to the function of another gene in E. coli constitute predicted ‘direct’ target-replacer gene pairs (via PROPER). We also predict ‘indirect’ target-replacer pairs where a replacer bypasses the target’s function (via GEM-PROPER). (4) Promiscuous activity of a ‘replacer’ gene can be confirmed for target-replacer pairs in which the target is conditionally essential on a minimal medium, via the multicopy suppression assay.

Proposed novel pathway for promiscuous production of pyridoxal 5’-phosphate.

GEM-PROPER was used to predict the indirect target-replacer pair, ∆pdxB/thiG, which we then confirmed with experiments. The predicted secondary function of thiG is pyridoxal 5’-phosphate synthase (P5PS), which would bypass the known 6-enzymatic-step pathway for production of p5p in E. coli. (a) The two alternative pathways, along with known promiscuous pathways in E. coli for producing p5p after pdxB knockout (as reported in Kim: []). Abbreviations are: ru5p-D = D-Ribulose 5-phosphate; gln-L = L-glutamine; g3p = Glyceraldehyde 3-phosphate; glu-L = L-glutamate; Pi = Phosphate. (b) Structural alignment of a homology model of thiG (for E. coli, based on crystal structure of thiG from B. subtilis) with a crystal structure of B. subtilis pdxS, the gene that (in complex with another gene, pdxT) performs the P5PS function in B. subtilis. The proteins share the TIM barrel fold. (c) Close-up of the structural alignment in (b), focused on the active site of pdxS and the residues of thiG that we propose perform the pdxS function. The location of the close-up is shown with a box in (b).